Flow cytometry is a method of ascertaining components or structural features of cells or other particles, typically by optical means.
Conventional flow cytometers have been commercially available since the early 1970s and presently cost, for example, more than $120,000. They can be behemoths in size, occupying upwards of 13 cubic feet and weighing well over 200 pounds.
In conventional flow cytometers, as shown in FIGS. 1 and 2, sample fluid containing sample cells or microspheres having reactants on their surfaces is introduced from a sample tube into the center of a stream of sheath fluid. The sheath fluid is pumped much more quickly than the sample so that the cells or microspheres are constrained to the center of the sheath fluid. This process, known as hydrodynamic focusing, allows the cells to be delivered reproducibly to the center of the measuring point. Typically, the cells or microspheres are in single unit suspension in the flow cell.
A continuous wave laser 200 focuses a laser beam on them as they pass single-file through the laser beam by a continuous flow of a fine stream of the suspension. Lasers in conventional flow cytometers may require shaping a round beam into an elliptical beam to be focussed on the flow cell. As shown in FIG. 2, this elliptical beam may be formed from the round beam using a beam shaping prismatic expander 260 located between the laser and the flow cell.
When the objects of interest in the flow stream are struck by the laser beam, certain signals are picked up by detectors. These signals include forward light scatter intensity and side light scatter intensity. In the flow cytometers, as shown in FIGS. 1 and 2, light scatter detectors 230, 232 are usually located opposite the laser (relative to the cell) to measure forward light scatter intensity, and to one side of the laser, aligned with the fluid-flow/laser beam intersection to measure side scatter light intensity.
In front of the forward light scatter detector 230 can be an opaque bar 220 that blocks incident light from the laser. Forward light scatter intensity provides information concerning the size of individual cells, whereas side light scatter intensity provides information regarding the relative granularity of individual cells.
Known flow cytometers, such as disclosed in U.S. Pat. No. 4,284,412 to HANSEN et al., have been used, for example, to automatically identify subclasses of blood cells. The identification was based on antigenic determinants on the cell surface which react to antibodies which fluoresce. The sample is illuminated by a focused coherent light and forward light scatter, right angle light scatter, and fluorescence are detected and used to identify the cells.
As described in U.S. Pat. No. 5,747,349 to VAN DEN ENGH et al., some such flow cytometers use fluorescent microspheres, which are beads impregnated with a fluorescent dye. Surfaces of the microspheres are coated with a tag that is attracted to a receptor on a cell, an antigen, an antibody, or the like in the sample fluid. So, the microspheres, having fluorescent dyes, bind specifically to cellular constituents. Often two or more dyes are used simultaneously, each dye being responsible for detecting a specific condition.
Typically, the dye is excited by the laser beam from a continuous wave laser 200, and then emits light at a longer wavelength. As shown in FIG. 1, dichroic filters 240 split this emitted light and direct it through optical detectors 250, 252, 254 that can be arranged sequentially 90.degree. relative to the laser. The optical detectors 250, 252, 254 measure the intensity of the wavelength passed through a respective filter. The fluorescence intensity is a function of the number of fluorescent beads attached to a cell or aggregated at portions of the sample/sheath fluid.
FIG. 2 depicts a prior art flow cytometer which uses beam splitters 242, 244, 246 to direct light from the flow cell 210 to photo-multiplier and filter sets 256, 258, 259 and to side light scatter detector 232. This flow cytometer employs a mirror 270 to reflect forward light scatter to forward light scatter detector 230.
If a specific wavelength could be separated out by each optical detector, multiple fluorescent dyes theoretically ought to be able to be applied to a single sample of cells or microspheres. However, I have determined that the properties of the fluorescent dyes themselves limit this flow cytometric technique to about three different wavelengths. The difference in energy, and hence wavelength, between an excitation photon and emission photon is known as Stokes shift. Generally, the larger the Stokes shift from the excitation wavelength, the broader and weaker the emission spectra.
At any given excitation wavelength, I have determined that there are often only about two or three commercially available dyes that emit a spectrum of wavelengths narrow enough and sufficiently separated enough that they are individually measurable simultaneously. Consequently, researchers in flow cytometry and other fields may have been limited to roughly three fluorescent labels, namely, for green, yellow-orange, and red light.
The limitation on the number of fluorescent labels necessarily crimps the amount of analysis that can be done on any one sample. Therefore, for meaningful analysis, a larger quantity of sample is required and more runs of the sample through the flow cytometer must be performed. This necessarily increases the time needed to analyze the sample. However, time is often not available in an emergency room environment, for example, where a small blood sample must be screened simultaneously for many diagnostic indicators, including therapeutic and abused drugs, hormones, markers of heart attack and inflammation, and markers of hepatic and renal function. In addition, for efficiency reasons, it is desirable to minimize the testing time to increase the number of tests that may be performed over a predetermined time interval.
One way to overcome the limitation on the number of fluorescent labels would be to use two lasers of different frequencies, each focused on a different spot along the flow stream. As a particle passes a first laser, up to three fluorescence measurements could be taken. Then, as the particle passes the second laser, theoretically, up to three more measurements could be taken. FIG. 3 illustrates this method.
It should be noted that the upper pair of particles A, B show the lower pair of particles A, B at a later time as the particles progress upward through the flow cell; the particles themselves are the same. In this case, laser #1 strikes particle A. Laser #2 must wait to emit its beam until it is believed that the same particle A is in range. Only then can the laser #2 emit its beam to strike particle A.
However, despite the intuitive simplicity of this dual laser approach, I have determined that it is often impossible to know for certain whether the measurements are made on the same particle. Because the measurement events at the sets of detectors are separated temporally and spatially, I have discovered that, besides laser emission timing problems, even the slightest flow turbulence can mix particles in suspension, thereby increasing the likelihood that subsequent measurements are not made on the same particle as the previous measurements.
For example, referring to FIG. 3, flow turbulence may cause particle B to change places with particle A such that laser #2 strikes particle B, instead of particle A. By extension, this unacceptable problem compounds as lasers and detectors are added to the device.
In view of this problem, I have determined that it would be desirable to have a system and/or a method for exciting a sample particle at multiple wavelengths in substantially the same time and space domain.
I have further determined that it would be desirable to extend such a system and such a method to a multitude of excitation wavelengths at no significant and/or measurable cost to measurement accuracy.
I have also determined that it would be desirable to have a system and/or a method for measuring multiple fluorescence wavelengths substantially simultaneously that is small and inexpensive relative to conventional flow cytometers.